In the proposed study entitled, "Testosterone stimulation of insulin signaling in adipocytes", we will investigate the molecular mechanisms of androgen modulation of the insulin signaling pathway promoting insulin sensitivity in fat cells. This research project on Diabetes 2 is a translational project that aims to improve our understanding and treatment of obesity and diabetes 2, both serious health problems which have a disproportionate impact on our minority community here in Los Angeles. Type 2 diabetes mellitus is associated with a metabolic syndrome of disturbances in lipid and carbohydrate metabolism including obesity, high blood glucose, and insulin resistance. This is strongly linked with increased cardiovascular disease, atherosclerosis, strokes, heart failure, and other serious effects on the health of men and women particularly in low-income and minority groups in Western societies. In diabetic patients with insulin resistance, the effects of testosterone (T) administration are poorly understood. In men, lower testosterone in serum is associated with type 2 diabetes mellitus 1, while T treatment stimulates increased insulin sensitivity and glucose uptake into cells, but a decrease in blood glucose, cholesterol and fat mass 2. The purpose of this study is to determine testosterone's effect on glucose uptake in differentiated adipocytes and to measure effects of T activation of the androgen receptor (AR) on modulating the insulin signaling pathway. Insulin is known to stimulate glucose uptake into adipocytes and myocytes or muscle cells by a multiple-step pathway involving the hormone insulin binding to the insulin receptor on the cell membrane and subsequent activation by phosphorylation of signaling molecules IRS-1 (insulin receptor substrate 1), PI3K (phospho-inositol triphosphate kinase), Akt (protein kinase B), AS160 (Akt substrate of 160 kD), and finally the active transport to the cell membrane of vesicles with GLUT4 protein (glucose transporter 4), that imports glucose into the interior of the muscle or fat cells. Using the 3T3-L1 cell line, our laboratory has shown that testosterone can inhibit the differentiation of adipocytes by about twofold by inhibition of the adipogenic transcription factors PPAR-gamma and C/EBP-1, mediated by AR binding to 2-catenin, which translocates to the nucleus to activate expression of transcription factor TCF/LEF 3. We recently found that testosterone treatment of mature 3T3-L1 adipocytes stimulates glucose uptake by over 50% within one to six hours, associated with an increase in phosphorylation and activation of Akt in the insulin signaling pathway, consistent with increased insulin sensitivity in men with diabetes 2. We hypothesize that testosterone stimulates glucose uptake in adipocytes by up-regulating insulin signaling through Akt, dependent on the androgen receptor. We further hypothesize that the mechanism for T stimulation of glucose uptake is to increase the transport of glucose into the cell by activating the GLUT4 glucose transporter protein to increase GLUT4 internal vesicle fusion with the cellular membrane, through activation of insulin signaling by increased phosphorylation of Akt and AS160. Our hypothesis will be tested by the following specific aims: Specific Aim 1: To investigate whether testosterone stimulation of glucose uptake in adipocytes is mediated by androgen receptor modulation of insulin signaling pathway. We will test if testosterone activation of androgen receptor stimulates glucose uptake in 3T3-L1 adipocytes by using the specific AR antagonists flutamide or bicalutamide to inhibit AR activation during the glucose uptake assay. We will test if the insulin signaling pathway through Akt mediates this effect by blocking insulin signaling with the specific PI3K inhibitors LY294002 and Wortmannin, which are known to block phosphorylation of Akt. Specific Aim 2: To investigate the specific mechanism of androgen modulation of glucose uptake by adipocytes. Assuming that Aim 1 is correct and insulin signaling is modulated by testosterone, we will investigate testosterone's effects on both the protein levels, and active phosphorylation states of these proteins in the insulin pathway: IRS-1 and phospho-IRS-1 (Ser307), Akt and phospho-Akt (Thr 308), AS160 and phospho-AS160 (Thr642). Finally, we will measure whether T stimulates the fusion of GLUT4 vesicles with the cellular membrane, promoting increased glucose uptake into the cell. Public Health Relevance: Understanding the molecular mechanisms of testosterone modulation of insulin signaling in adipocytes will allow us to see exactly which signaling molecules are involved in stimulating insulin sensitivity and glucose uptake into fat cells. It is necessary to investigate how the insulin signaling pathway normally works, in its interaction and cross-talk with the androgen signaling pathway, in order to utilize these pathways to devise more effective therapies for Type 2 diabetes. This understanding will hopefully lead to the development of new strategies to prevent diabetes progression into life-threatening diseases such as coronary artery disease, strokes, or myocardial infarction.